Participação do Sistema Endocanabinoide na Inibição da Secreção Salivar

Prévia do material em texto

Participation of the endocannabinoid system in
lipopolysaccharide-induced inhibition of salivary secretion
Javier Fernandez-Solari a,b,*, Juan Pablo Prestifilippo a, Cesar Angel Ossola a,
Valeria Rettori b, Juan Carlos Elverdin a
aCátedra de Fisiologı́a, Facultad de Odontologı́a, Universidad de Buenos Aires, Argentina
bCentro de Estudios Farmacológicos y Botánicos (CEFyBO-CONICET-UBA), Argentina
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0
a r t i c l e i n f o
Article history:
Accepted 15 May 2010
Keywords:
Endocannabinoid system
Submandibular gland
Lipopolysaccharide
TNFa
AM251
AM630
a b s t r a c t
Objective: The aim of the present paper was to assess whether lipopolysaccharide (LPS)-
induced inhibition of salivary secretion involves the activation of the endocannabinoid
system and the participation of tumor necrosis factor (TNF)a in the submandibular gland.
Design: Pharmacological approaches were performed by using CB1 and/or CB2 cannabinoid
receptor antagonists, AM251 and AM630, respectively, injected into the submandibular
gland, to study the participation of the endocannabinoid system in LPS inhibitory effects on
metacholine-induced salivary secretion. To assess the participation of TNFa on LPS inhibi-
tory effects, salivary secretion was studied in LPS treated rats after the intraglandular
injection of etanercept, a soluble form of TNF receptor which blocks TNFa action. Finally, to
evaluate the possible interplay between endocannabinoids and TNFa on the submandibular
gland function reduced during LPS challenge, the salivary secretion was studied after the
intraglandular injection of this cytokine alone or concomitantly with AM251 and AM630.
Results: AM251 and AM630, injected separately or concomitantly, partially prevented LPS-
induced inhibition of salivation. Also, anandamide synthase activity was increased in
submandibular glands extracted from rats 3 h after LPS injection, suggesting that the
endocannabinoid system was activated in response to this challenge. On the other hand,
etanercept, prevented the inhibitory effect of LPS on salivary secretion and moreover, TNFa
injected intraglandularly inhibited salivary secretion, being this effect prevented by AM251
and AM630 injected concomitantly.
Conclusion: The present results demonstrate the participation of the endocannabinoid
system and TNFa on salivary responses during systemic inflammation induced by LPS.
# 2010 Elsevier Ltd. All rights reserved.
avai lab le at www.sc iencedi rect .com
journal homepage: http://www.elsevier.com/locate/aob
1. Introduction
Lipopolysaccharide (LPS), an integral part of the outer
membrane of gram-negative bacteria, is the main pathogen-
ic factor that leads to endotoxemia. Macrophages are the
primary target for LPS, where the endotoxin interacts with
the CD14 protein/toll-like receptor-4 complex to activate
* Corresponding author at: Centro de Estudios Farmacológicos y Botán
Aires, Argentina. Tel.: +54 1145083680.
E-mail address: javierfsolari@yahoo.com.ar (J. Fernandez-Solari).
0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserve
doi:10.1016/j.archoralbio.2010.05.006
multiple signalling pathways.1,2 This signalling pathways
lead to the activation of a variety of transcription factors,3
that regulate gene expression encoding inflammatory
mediators.4 Lipopolysaccharide induces the expression of
cytokines such as tumor necrosis factor alpha (TNFa),
interleukin-1 (IL-1), IL-6, and IL-8, which have been
implicated in the pathophysiology of sepsis.1 Additionally,
icos (CEFyBO-CONICET-UBA), Paraguay 2155 (C1121ABG), Buenos
d.
mailto:javierfsolari@yahoo.com.ar
http://dx.doi.org/10.1016/j.archoralbio.2010.05.006
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0584
LPS induces the production of different lipid mediators in
macrophages, such as prostaglandins,5 leukotrienes6 and
endocannabinoids.7 The best-known endocannabinoids are
arachidonoyl ethanolamide (anandamide) and arachidonoyl
glycerol, both derivatives of arachidonic acid.8 Mammalian
tissues contain, at least two types of cannabinoid receptors,
CB1 and CB2, concentrated in the nervous system
9–11 and
peripheral tissues, respectively.12 Both receptors are coupled
to Gi/o proteins and respond by inhibiting the activity of
adenylyl cyclase.13
The submandibular gland is one of the major salivary
glands, together with the sublingual and the parotid glands.
End secretory units, called acini, are continuous with a
ductal system that leads the saliva to the oral cavity.14
Previously, we have described the presence of CB1 and CB2
receptors in the ductal system of the submandibular gland
and, additionally, the expression of CB2 receptors in the
periphery of acinar cells.15 We also have shown that
anandamide (10 ng/50 ml) injected into the submandibular
gland decreases salivary secretion through the activation of
both cannabinoid receptors, since AM251 and AM630, CB1
and CB2 receptor antagonists respectively, block this
inhibitory effect.15
Salivary secretion is altered in different pathological states.
Moreover, we have previously demonstrated that LPS (5 mg/
kg/3 h) injected intraperitoneally inhibits salivary secretion by
increasing the production of prostaglandins, that are deriva-
tives of arachidonic acid like endocannabinoids.16 Also, TNFa
is known to be released after LPS administration and mediates
a number of effects attributed to LPS; therefore it could be
involved in LPS-induced inhibition of salivary secretion. In
addition, anandamide content is rapidly increased in different
tissues in response to LPS intraperitoneal or intravenous
injection.7,17 Furthermore, anandamide is able to inhibit
proinflammatory cytokines production, including TNFa in
LPS-stimulated monocytes18 and rat microglial cells,19 sug-
gesting that endocannabinoids modulate inflammatory
responses.
Based in the evidences described, the aim of the present
work was to assess whether LPS-induced inhibition of
salivary secretion involves the participation of TNFa and
the activation of the endocannabinoid system in the
submandibular gland.
2. Materials and methods
2.1. Chemicals
Anandamide, Forskolin and LPS from Escherichia coli were
purchased from Sigma Chemicals (St. Louis, MO, USA).
Chloralose and methacholine were obtained from FLUKA
(Laborchemikalien, Berlin, Germany). TNFa was purchased
from Promega Corporation (Madison, WI, USA). AM251 [N-
(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide] and AM630 6-Iodo-2-
methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxy-
phenyl) methanone were obtained from TocrisTM (Ellisville,
MO, USA). Etanercept was purchased from Amgem and Wyeth
Pharmaceuticals (Philadelphia; USA).
2.2. Animals
Adult male Wistar rats (250–300 g) from our own colony
(Department of Biochemistry, Dental School, University of
Buenos Aires) were kept in group cages in an animal room
having a photoperiod of 12 h of light (07:00–19:00), room
temperature of 22–25 8C and free access to rat chow and tap
water. The animals were divided into several experimental
groups (6–8 animals/group). The experimental procedures
reported here were approved by the Animal Care Committee
of the Center for Pharmacological and Botanicals Studies of
the National Council of Scientific and Technical Research of
Argentina and were carried out in accordance with the
National Institute of Health of USA guidelines.
2.3. ‘‘In vivo’’ studies
2.3.1. Salivary secretion studies
Salivary responses were determined in anesthetized rats
(chloralose 100 mg/kg, 0.5 ml NaCl 0.9% iv). The submandibu-
lar ducts were cannulated with a fine glass cannula, and
salivary secretion was induced by different doses of metha-
choline (1, 3 and 10 mg/kg in saline) administered sequentially
via the right femoral vein. The saliva was collected, for 3 min
after the injection, on aluminumfoil and weighed as
previously described.20 Resting flow of saliva (unstimulated)
was not observed in rats. There were 5–6 rats per group and
results were expressed as mg of saliva/3 min.
In the first group of experiments, the rats received
intraperitoneal injections of LPS (5 mg/kg) or saline as vehicle.
To evaluate the participation of the endocannabinoid system
in salivary responses to LPS, AM251 (15 mg in 50 ml of 1%
dimethyl sulfoxide), a selective antagonist for CB1 receptors,
21
AM630 (15 mg in 50 ml of 1% dimethyl sulfoxide), a selective
antagonist for CB2 receptors
22 or vehicle were injected into the
submandibular gland. The injections were performed with a
30 G � needle and the substances were injected very slowly
between the capsule that covers the gland and the parenchy-
ma. In order to be sure that the substances reached the entire
gland, the injections were performed at 30 min, 1.5 and 2.5 h
post-LPS injection. Three hours after LPS injection, dose–
response curves to methacholine were performed to evaluate
salivary secretion. The doses of cannabinoid receptor antago-
nists employed were obtained from our previous reports.15,23
To assess whether TNFa is involved in LPS-induced
inhibition of salivary secretion, we injected etanercept
(800 mg in 50 ml), a TNFa antagonist, or saline as vehicle, into
the submandibular gland 2.5 h after LPS intraperitoneal
injection and 30 min after, dose–response curves to metha-
choline on salivary secretion were performed.
Therefore, the rats were divided in six groups: (1) vehicle,
(2) LPS (5 mg/kg, intraperitoneal), (3) LPS + AM630 (15 mg/50 ml,
intraglandular), (4) LPS + AM251 (15 mg/50 ml, intraglandular),
(5) LPS + AM630 + AM251 and (6) LPS + etanercept (800 mg/
50 ml, intraglandular).
Additionally, to confirm whether TNFa (300 ng in 50 ml,
intraglandular) alters salivary secretion by activating the
endocannabinoid system, we studied its effect on salivary
secretion when it was injected alone or 10 min after the
injection of AM251 (15 mg/50 ml) and AM630 (15 mg/50 ml) into
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0 585
the submandibular gland. Thirty minutes after TNFa injec-
tions, dose–response curves to methacholine on salivary
secretion were performed.
Additionally, 3 h after LPS injection (5 mg/kg, intraperito-
neal), submandibular glands were extracted from anesthe-
tized rats and homogenized in 1 ml of H2O and centrifuged at
6000 � g for 10 min at 4 8C. Supernatants were collected and
stored at�20 8C until cyclic adenosine monophosphate (cAMP)
measurements.
2.4. ‘‘In vitro’’ studies
The animals were killed by decapitation, and submandibular
glands were removed. Submandibular glands (5–6 per group)
were cut in halves to enhance the penetration of the different
substances into the tissue and preincubated in 500 ml of Krebs–
Ringer bicarbonate buffer medium (pH 7.4) containing 0.1%
glucose in a Dubnoff metabolic shaker (50 cycles per min, 95%
O2/5% CO2) for 15 min before replacement with fresh medium
containing the compounds to be tested. The incubation was
continued for another 30 min with forskolin (80 mM), an
adenylyl cyclase stimulator, forskolin plus TNFa
(3 � 10�9 M); forskolin plus TNFa plus AM251 (10 mM), AM251
alone; forskolin plus TNFa plus AM630 (10 mM); AM630 alone;
forskolin plus TNFa plus AM251 plus AM630; and finally,
AM251 plus AM630. After the incubations, the submandibular
glands were homogenized in 1 ml of distilled H2O and
centrifuged at 6000 � g for 10 min at 4 8C. Supernatants were
collected and stored at �20 8C until cAMP measurements.
2.5. Radioimmunoassay
cAMP was measured by radioimmunoassay, using the highly
specific cAMP antibody kindly provided by Dr. A.F. Parlow
(National Hormone & Peptide Program, CA, USA). The assay
sensitivity was 0.061 pmol/ml. Intra- and inter-assay coeffi-
cients of variation were 8.1 and 10.5%, respectively.
2.6. Anandamide production
The production of anandamide was assayed as described by
Paria et al. 1996 with minor modifications.24 The submandib-
ular glands were removed 3 h after the injection of LPS (5 mg/
kg, intraperitoneal), homogenized in 800 ml of buffer [20 mM
Tris–HCl/1 mM EDTA (pH 7.6)] and centrifuged at 2000 � g for
15 min. Supernatant protein (150–100 mg) was incubated in a
total volume of 200 ml of 50 mM Tris–HCl (pH 9.0) with 40 mM
(0.1 mCi) of [1-14C]arachidonic acid (40–60 mCi/mmol, PerkinEl-
mer, Waltham, MA, USA) and 20 mM ethanolamine (Sigma
Chemicals, St. Louis, MO, USA) during 5 min at 37 8C. The
reaction was terminated by the addition of 400 ml of chloro-
form–methanol (1:1) mixture. Two additional washes of the
aqueous phase with 400 ml of chloroform were performed.
Organic phases were evaporated to dryness under nitrogen
gas and dissolved in 40 ml of chloroform–methanol (1:1).
Samples and standards were applied on Silica Gel 60 plates
(Merck, Darmstadt, Germany). The synthesized [14C]ananda-
mide was resolved by using the organic layer of an ethyl
acetate–hexane–acetic acid–water (100:50:20:100) mixture.
The plate was exposed to X-ray film at �70 8C. After
autoradiography, distribution of radioactivity on the plate
was counted in a scintillation counter by scraping off the
corresponding spots of the plate. The retardation factor values
of anandamide and arachidonic acid were 0.33 and 0.78,
respectively.
2.7. Determination of TNFa
TNFa was determined by the sandwich enzyme immunoassay
technique using a rat TNFa Kit provided by Quantikine M, R&D
Systems (Minneapolis, MN, USA). The optical density was
determined at 450 nm with a wavelength correction set at 540
using a microplate reader (Bio-Rad model 550). These assays
have been shown to be specific for rat TNFa. The sensitivity of
the assay was 12.5 pg/ml per tube and the curve was linear up
to 400 pg/ml of TNFa. The inter- and intra-assay variation was
3.5 and 9.4%, respectively.
2.8. Statistics
Data are presented as the mean � SE. Comparisons between
more than two groups were performed by one-way analysis of
variance (ANOVA) followed by the Student–Newman–Keuls
multiple comparison test or by two-way ANOVA followed by
the Bonferroni post-test. When two groups were compared,
Student’s t-test was used. All analyses were performed with
the Graph-Pad Instat software. Differences with p values<0.05
were considered statistically significant.
3. Results
3.1. Effect of cannabinoid receptor antagonists and
etanercept on LPS-induced inhibition of salivary secretion
Since the inhibitory actions of LPS and anandamide on
salivary secretion are similar, we hypothesized that the effect
of LPS might be mediated, at least in part, by the endocanna-
binoid system. For that reason, we assessed the effect of
selective antagonists for CB1 and CB2, AM251 and AM630,
respectively, on salivary secretion reduced by LPS. The
inhibitory effect of LPS (5 mg/kg/3 h) on methacholine (3
and 10 mg/kg)-induced salivary secretion was prevented by
intraglandular injections of AM251 (15 mg/50 ml) at 3 mg/kg of
methacholine and partially blocked at 10 mg/kg of methacho-
line (P < 0.01 vs. vehicle) (Fig. 1). Additionally, the inhibitory
effect of LPS on methacholine-induced salivary secretion was
partially blocked by injecting AM630 (15 mg/50 ml) at 10 mg/kg of
methacholine (P < 0.001 vs. vehicle). Furthermore, the simul-
taneous injection of both antagonists, AM251 and AM630,
completely prevented the inhibitory effect of LPS on salivary
secretion induced by 3 mg/kg of methacholine and partially
blocked it at 10 mg/kg of methacholine (P < 0.05 vs. vehicle).
Since TNFa is one of the well-known mediators of LPS
actions, we studied LPS effect on salivary secretion after
intraglandular injection of etanercept, a TNFa antagonist.
Etanercept (800 mg/50 ml) prevented completely LPS inhibitory
effect on salivary secretion induced by 3 mg/kg of methacho-
line and partially when 10 mg/kg of methacholinewas used
(P < 0.001 vs. vehicle) (Fig. 1).
Fig. 1 – Effect of intraglandular injection of AM251
(15 mg/50 ml), AM630 (15 mg/50 ml), AM251 plus AM630 and
etanercept (800 mg/50 ml) on LPS (5 mg/kg/3 h,
intraperitoneal)-induced inhibition on methacholine-
stimulated salivary secretion. Values are mean W SE (5–6
rats per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle.
#P < 0.05 and ###P < 0.001 vs. LPS and +P < 0.05 vs. LPS plus
AM630 (two-way ANOVA followed by Bonferroni post-
test).
Fig. 2 – Effect of LPS (5 mg/kg/3 h, ip) on (A) cyclic AMP
content and (B) production of AEA in the submandibular
gland (SMG). Values are means W SE (7–8 rats per group).
*P < 0.05 and **P < 0.01 vs. vehicle (Student’s t-test).
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0586
3.2. Effect of LPS administration on cAMP content and
anandamide production in the submandibular gland
Lipopolysaccharide (5 mg/kg/3 h) injected intraperitoneally
decreased significantly (P < 0.05) cAMP content in the sub-
mandibular gland. This effect could be due to activation of Gi
coupled cannabinoid receptors by increased levels of endo-
cannabinoids (Fig. 2A). To confirm this hypothesis, we studied
the effect of LPS injected intraperitoneally on anandamide
production in submandibular glands ex vivo. Fig. 2B shows that
LPS, 3 h after its injection, increased significantly (P < 0.01)
anandamide production.
3.3. Effect of LPS on TNFa plasma levels and content in the
submandibular gland
Since it is well known that LPS stimulates TNFa production, we
studied the in vivo effect of LPS on TNFa plasma concentration
and content in the submandibular gland. Lipopolysaccharide
(5 mg/kg/, intraperitoneal) increased TNFa plasma concentra-
tion 30 min after its injection, showing a peak at 1 and 3 h post-
injection (Fig. 3A). Also, TNFa content was increased in
submandibular glands 3 and 6 h after LPS injection as
compared to control values (Fig. 3B).
3.4. Inhibitory effect of TNFa on cAMP content in
submandibular glands in vitro
Cyclic AMP content was increased (P < 0.01) by forskolin
(8 � 10�5 M), an activator of AC, in submandibular glands in
vitro (Fig. 4). This increase in cAMP was significantly blocked
(P < 0.01) by TNFa (3 � 10�9 M). To investigate whether the
inhibition of TNFa on cAMP content is mediated by cannabi-
noid receptors, AM251 (10�5 M) and AM630 (10�5 M) were
added to the media together with TNFa and forskolin or alone.
AM251 as well as AM630 blocked (P < 0.05) the inhibition of
TNFa on forskolin stimulated cAMP content. When AM251 and
AM630 were used together, the blockade of TNFa inhibitory
effect was higher (P < 0.01). Neither AM251 nor AM630 alone
nor AM251 and AM630 together had any effect on cAMP
content as compared to control values as can be seen in Fig. 4.
3.5. Effect of TNFa and cannabinoid receptor antagonists
on salivary secretion
To ensure that TNFa mediates LPS-induced inhibition of
salivary secretion by activating the endocannabinoid system
we studied the effect of intraglandular injections of TNFa and
cannabinoid receptor antagonists on salivary secretion. We
observed that TNFa (300 ng/50 ml) inhibited methacholine-
induced salivary secretion (P < 0.05 at 1 mg/kg and P < 0.001 at
3 and 10 mg/kg vs. vehicle) and that previous intraglandular
injection of AM251 (15 mg/50 ml, ig) plus AM630 (15 mg/50 ml)
prevented almost totally this inhibitory effect (Fig. 5).
4. Discussion
During the last years the number of studies on the effects of
endocannabinoids in physiological and pathophysiological
conditions has been increased. Moreover, the participation of
endocannabinoids in the cytokine network of various organs
Fig. 3 – Effect of LPS (5 mg/kg, ip) on time course of TNFa
concentration in (A) plasma and (B) submandibular gland
(SMG). Values are means W SE (6 rats per group). *P < 0.05
vs. 30 min and 6 h (one-way ANOVA followed by Student–
Newman–Keuls multiple comparison test).
Fig. 4 – Effect of TNFa (3 T 10S9 M), AM251 (10S5 M), AM630
(10S5 M) and the combinations of them on cAMP content
increased by forskolin in submandibular glands (SMG) in
vitro. Values are means W SE (6 rats per group). **P < 0.01
and ***P < 0.001 vs. control. ##P < 0.01 vs. forskolin.
sP < 0.05 and ssP < 0.01 vs. forskolin + TNFa (one-way
ANOVA followed by Student–Newman–Keuls multiple
comparison test).
Fig. 5 – Effect of intraglandular injection of TNFa (300 ng/
50 ml) alone and after the injection of AM251 (15 mg/50 ml)
plus AM630 (15 mg/50 ml) on methacholine-stimulated
salivary secretion. Values are mean W SE (5–6 rats per
group). *P < 0.05 and ***P < 0.001 vs. vehicle. ###P < 0.001 vs.
TNFa (two-way ANOVA followed by Bonferroni post-test).
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0 587
and tissues was reported and its therapeutic potential was
proposed.25,26
In the present work, we demonstrate that LPS injected
intraperitoneally reduced salivary secretion 3 h after its
injection and also increased anandamide production and
decreased cAMP content, a second messenger whose reduc-
tion is linked to cannabinoid receptors activation,13 in the
submandibular gland. In concordance, other authors reported
that LPS stimulates anandamide production in human
peripheral lymphocytes and mouse macrophages.7,27 Differ-
ent hypotheses were proposed about the increases of
anandamide in response to LPS: (1) down-regulation of fatty
acid amide hydrolase expression in immune cells,27 the
enzyme implicated in anandamide degradation; (2) direct
increase of anandamide production through a phospholipase
C/phosphatase pathway.7 In addition, we have previously
reported that LPS-induced inhibition of salivary secretion is
associated to the increase of prostaglandin E2, another
derivative of arachidonic acid.16 Therefore, we hypothesized
that the endocannabinoid system could mediate the inhibito-
ry effect of LPS on salivary secretion. The present results
demonstrate that both CB1 and CB2 antagonists blocked, at
least partially, the inhibitory effect of LPS on methacholine-
induced salivary secretion when were injected intraglandu-
larly. This effect may be due to an increase in the availability of
cAMP by the blockade of cannabinoid receptors, probably
resulting in elevated cytosolic calcium levels and the conse-
quent high salivary flow in response to methacholine. As was
mentioned in introduction, cannabinoid receptors are mainly
coupled to Gi/o proteins and their activation produce an
inhibitory effect of adenylyl cyclase activity and the conse-
quent decrease of cAMP production.13 Although calcium is the
main second messenger that mediates salivary secretion after
muscarinic cholinergic receptor activation, an emerging body
of evidence indicates that, a cross-talk exist between
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0588
phospholipase C/inositol triphosphate/calcium and adenylyl
cyclase/cAMP pathways that regulates the calcium transport
processes.28,29 Moreover, it was suggested that adenylyl
cyclase/cAMP is the intracellular signalling pathway resulting
in elevated cytosolic calcium levels. In fact, acetylcholine-
evoked secretion from the parotid gland is substantially
enhanced by cAMP-raising agonist.30
Cannabinoid receptors are expressed in different struc-
tures of the submandibular gland such as acini, ducts as well
as nervous terminals15 and their activation could probably
inhibit the release of neurotransmitters from pre-synaptic
terminals on the submandibular gland, consequently inhibit-
ing salivation. In this line, it was reported that D9-tetrahydro-
cannabinol administrated through the femoral vein decreased
salivary flow from submandibular gland during electrical
stimulation by a mechanism involving a decrease in the
release of acetylcholine.31
It is well documented that LPS increases the plasma
concentration of different cytokines includingTNFa, one of
the most important cytokine involved in the pathophysiology
of sepsis.1 Moreover, it was reported that the proinflammatory
cytokines IL-1b, IL-6 and TNFa were induced by LPS via toll-
like receptor type 4 in the submandibular gland of mice.32
Particularly, TNFa has been associated with alterations of
salivary secretion flow and composition.33,34 Here we show
that etanercept, a dimeric soluble form of the 75-kDa TNF
receptor, injected intraglandularly, prevented the inhibitory
effect of LPS on salivary secretion. This result confirmed that
LPS inhibitory effect was mediated, at least partially, by TNFa.
The anti-inflammatory effects of etanercept are due to its
ability to bind to TNF, preventing its interaction with cell-
surface receptors and rendering it biologically inactive.35
To investigate the possible role of TNFa in LPS induced
activation of the endocannabinoid system in the submandib-
ular gland, we first evaluated TNFa plasma concentration and
content in the submandibular gland after LPS injection. We
demonstrated that LPS not only increased TNFa plasma
concentration from 30 min exhibiting a higher response at
1 h, but also increased TNFa content in the submandibular
gland from 3 h post-LPS injection. Additionally, we showed
that TNFa injected intraglandularly inhibited methacholine-
induced salivary secretion. These results are supported by
previous work showing that TNFa production is an important
contributor to secretory dysfunction in Sjögren’s syndrome by
disrupting salivary epithelial cell functions necessary for
saliva secretion.33 Also, TNFa-induced inhibition of salivary
secretion was prevented by AM251/AM630 injected 10 min
before by the same route, confirming the participation of the
endocannabinoid system in the salivation altered during the
inflammatory state.
To further confirm the participation of the endocannabi-
noid system in TNFa-induced inhibition of salivary secretion,
we measured the in vitro effect of TNFa on forskolin induced
increase in cAMP content in submandibular glands incubated
with or without cannabinoid receptor antagonists. We
demonstrated that AM251 and AM630 blocked the inhibitory
effect of TNFa on forskolin induced cAMP production as
anandamide does in similar experimental conditions.15
Therefore, the increase of anandamide after LPS injection in
the submandibular gland could probably be due to the
proinflammatory state produced by cytokines. It was also
reported that anandamide could generate a negative feedback
exerting inhibitory effects on proinflamatory cytokine pro-
duction, confirming its role as a modulatory agent.18,36,37 Also,
there is evidence that in inflammatory conditions, cannabi-
noid receptor agonists down-regulate mast cells and granu-
locytes, and reduce cytokine release by acting on CB1 and CB2
receptors.38–40
On the other hand, since LPS injected intraperitoneally as
well as TNFa injected intracerebroventricularly were shown to
increase anandamide in the hypothalamus,17,41 an area that
has been shown to regulate autonomic inputs to the salivary
glands via endocannabinoids,42,43 we cannot discard the
participation of the hypothalamic endocannabinoid system
in LPS inhibitory effects of salivary secretion.
In summary, the present results show that the inflamma-
tory state induced by LPS decreased methacholine-induced
salivary secretion by increasing TNFa and anandamide that
activates cannabinoid receptors. The participation of the
endocannabinoid system during the inflammatory state
involves both CB1 and CB2 receptors which activate signal
transduction pathways whose final biological signal is the
reduction of salivary secretion. Here, we report for the first
time the participation of the endocannabinoid system on
salivary responses during systemic inflammation indicating
that cannabinoid receptors could be potential therapeutic
targets for the treatment of secretory dysfunctions such as
hyposialia.
Acknowledgements
This work was supported by grants from the University of
Buenos Aires (UBACyT O 007), Agencia Nacional de Promoción
Cientı́fica Tecnológica (Grants Prestamos BID PICT 07-1016 and
06-0258), Fundación Alberto J. Roemmers and the National
Council of Scientific a Technical Research of Argentina
(CONICET). The authors are greatly indebted to Ricardo
Horacio Orzuza for technical assistance.
Funding: Financial support by National Agency of Scientific
and Technical Promotion from Argentina PICT 07-1016.
Financial support by Roemmers Foundation. Financial support
by UBACyT project from the University of Buenos Aires,
Argentina.
Competing interests: None declared.
Ethical approval: Not required.
r e f e r e n c e s
1. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC.
CD14, a receptor for complexes of lipopolysaccharide (LPS)
and LPS binding protein. Science 1990;249:1431–3.
2. Dentener MA, Bazil V, Von Asmuth EJ, Ceska M, Buurman
WA. J Immunol 1993;150:2885–91.
3. Muller JM, Ziegler-Heitbrock HW, Bauerle PA. Nuclear factor
kappa B, a mediator of lipopolysaccharide effects.
Immunobiology 1993;187:233–56.
4. Guha M, Mackman N. LPS induction of gene expression in
human monocytes. Cell Signal 2001;13:85–94.
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0 589
5. Minghetti L, Levi G. Induction of prostanoid biosynthesis by
bacterial lipopolysaccharide and isoproterenol in rat
microglial cultures. J Neurochem 1995;65:2690–8.
6. Rankin JA, Sylvester I, Smith S, Yoshimura T, Leonard EJ.
Macrophages cultured in vitro release leukotriene B4 and
neutrophil attractant/activation protein (interleukin 8)
sequentially in response to stimulation with
lipopolysaccharide and zymosan. J Clin Invest 1990;86:
1556–64.
7. Liu J, Bátkai S, Pacher P, Harvey-White J, Wagner JA, Cravatt
BF, et al. Lipopolysaccharide induces anandamide synthesis
in macrophages via CD14/MAPK/phosphoinositide 3-kinase/
NF-kappaB independently of platelet-activating factor. J Biol
Chem 2003;278:45034–9.
8. Mechoulam R, Fride E, Di Marzo V. Endocannabinoids. Eur J
Pharmacol 1998;359:1–18.
9. Devane WA, Dysarz 3rd FA, Johnson MR, Melvin LS, Howlett
AC. Determination and characterization of a cannabinoid
receptor in rat brain. Mol Pharmacol 1988;34:605–13.
10. Herkenham M, Lynn AB, Little MD, Johnson MR, Melvin LS,
de Costa BR, et al. Cannabinoid receptor localization in
brain. Proc Natl Acad Sci USA 1990;87:1932–6.
11. Pertwee RG. In: Pertwee RG, editor. Cannabinoid receptors.
London: Academic Press; 1995. p. 1–34.
12. Munro S, Thomas KL, Abu-Shaar M. Molecular
characterization of a peripheral receptor for cannabinoids.
Nature 1993;365:61–5.
13. Howlett AC, Fleming RM. Cannabinoid inhibition of
adenylate cyclase. Pharmacology of the response in
neuroblastoma cell membranes. Mol Pharmacol 1984;26:
532–8.
14. Tandler B, Phillips CJ, Microstructure of mammalian
salivary glands and its relationship to diet.Garret JR,
Ekstrom J, Anderson LC, editors. Glandular mechanisms of
salivary secretion oral frontiers on biology, vol. 10. Basel,
Switzerland: Karger; 1998. p. 21–35.
15. Prestifilippo JP, Fernández-Solari J, de la Cal C, Iribarne M,
Suburo AM, Rettori V, et al. Inhibition of salivary secretion
by activation of cannabinoid receptors. Exp Biol Med
2006;231:1421–9.
16. Lomniczi AC, Mohn A, Faletti A, Franchi A, McCann SM,
Rettori V, et al. Inhibition of salivary secretion by
lipopolysaccharide: possible role of prostaglandins. Am J
Physiol Endocrinol Metab 2001;281:E405–11.
17. Fernandez-Solari J, Prestifilippo JP, Bornstein SR, McCann
SM, Rettori V. Participation of the endocannabinoid system
in the effect of TNFa on hypothalamic release of
gonadotropin-releasing hormone. Ann NY Acad Sci
2006;1088:238–50.
18. Berdyshev EV, Schmid PC, Krebsbach RJ, Schmid HH.
Activation of PAF receptors results in enhanced synthesis of
2-arachidonoylglycerol (2-AG) in immune cells. FASEB J
2001;15:2171–8.
19. Facchinetti F, Del Giudice E, FuregatoS, Passarotto M, Leon
A. Cannabinoids ablate release of TNFa in rat microglial
cells stimulated with lypopolysaccharide. Glia 2003;41:
161–8.
20. Bianciotti LG, Elverdin JC, Vatta MS, Colatrella C, Fernandez
BE. Atrial natriuretic factor enhances induced salivary
secretion in the rat. Regul Pept 1994;49:195–202.
21. Gatley SJ, Gifford AN, Volkow ND, Lan R, Makriyannis A.
123Ilabeled AM251: a radioiodinated ligand which binds in
vivo to mouse brain cannabinoid CB1 receptors. Eur J
Pharmacol 1996;307:331–8.
22. Pertwee R, Griffin G, Fernando S, Li X, Hill A, Makriyannis A.
AM630, a competitive cannabinoid receptor antagonist. Life
Sci 1995;56:1949–55.
23. Prestifilippo JP, Fernández-Solari J, Medina V, Rettori V,
Elverdin JC. Role of the endocannabinoid system in ethanol-
induced inhibition of salivary secretion. Alcohol Alcohol
2009;44:443–8.
24. Paria B, Deutsch D, Dey S. The uterus is a potential site for
anandamide synthesis and hydrolysis: differential profiles
of anandamide synthase and hydrolase activities in the
mouse uterus during the periimplantation period. Mol
Reprod Dev 1996;45:183–92.
25. Klein TW, Lane B, Newton CA, Friedman H. The cannabinoid
system and cytokine network. Proc Soc Exp Biol Med
2000;225:1–8.
26. Ramos JA, González S, Sagredo O, Gómez-Ruiz M,
Fernández-Ruiz J. Therapeutic potential of the
endocannabinoid system in the brain. Mini Rev Med Chem
2005;5:609–17.
27. Maccarrone M, De Petrocellis L, Bari M, Fezza F, Salvati S, Di
Marzo V, et al. Lipopolysaccharide downregulates fatty acid
amide hydrolase expression and increases anandamide
levels in human peripheral lymphocytes. Arch Biochem
Biophys 2001;393:321–8.
28. Bugrim AE. Regulation of Ca2+ release by cAMP-dependent
protein kinase. A mechanism for agonist-specific calcium
signaling? Cell Calcium 1999;25:219–26.
29. Zhang GH, Martinez JR. Effects of forksolin, dibutyryl cAMP
and H89 on Ca2+ mobilization in submandibular salivary
cells of newborn rats. Arch Oral Biol 1999;44:735–44.
30. Bruce JI, Shuttleworth TJ, Giovannucci DR, Yule DI.
Phosphorylation of inositol 1,4,5-trisphosphate receptors in
parotid acinar cells. A mechanism for the synergistic effects
of cAMP on Ca2+ signaling. J Biol Chem 2002;277:1340–8.
31. McConnell WR, Dewey WL, Harris LS, Borzelleca JF. A study
of the effect of delta 9-tetrahydrocannabinol (delta9-THC)
on mammalian salivary flow. J Pharmacol Exp Ther
1978;206:567–73.
32. Yao C, Li X, Murdiastuti K, Kosugi-Tanaka C, Akamatsu T,
Kanamori N, et al. Lipopolysaccharide-induced elevation
and secretion of interleukin-1b in the submandibular gland
of male mice. Immunology 2005;116:213–22.
33. Baker OJ, Camden JM, Redman RS, Jones JE, Seye CI, Erb L,
et al. Proinflammatory cytokines tumor necrosis factor-
alpha and interferon-gamma alter tight junction structure
and function in the rat parotid gland Par-C10 cell line. Am J
Physiol Cell Physiol 2008;295:C1191–201.
34. Jonsson MV, Delaleu N, Brokstad KA, Berggreen E, Skarstein
K. Impaired salivary gland function in NOD mice association
with changes in cytokine profile but not with
histopathologic changes in the salivary gland. Arthritis
Rheum 2006;54:2300–5.
35. Di Paola R, Mazzon E, Muià C, Crisafulli C, Terrana D, Greco
S, et al. Effects of etanercept, a tumour necrosis factor-alpha
antagonist, in an experimental model of periodontitis in
rats. Br J of Pharmacol 2007;150:286–97.
36. Iribarne M, Torbidoni V, Julián K, Prestifilippo JP, Sinha D,
Rettori V, et al. Cannabinoid receptors in conjunctival
epithelium: identification and functional properties. Invest
Ophthalmol Vis Sci 2008;49:4535–44.
37. Sancho R, Calzado MA, Di Marzo V, Appendino G, Muñoz E.
Anandamide inhibits nuclear factor-kappaB activation
through a cannabinoid receptor-independent pathway. Mol
Pharmacol 2003;63:429–38.
38. Richardson JD, Kilo S, Hargreaves KM. Cannabinoids reduce
hyperalgesia and inflammation via interaction with
peripheral CB1 receptors. Pain 1998;75:111–9.
39. Parolaro D. Presence and functional regulation of cannabinoid
receptors in immune cells. Life Sci 1999;65:637–44.
40. Sacerdote P, Massi P, Panerai AE, Parolaro D. In vivo and in
vitro treatment with the synthetic cannabinoid CP55,940
decreases the in vitro migration of macrophages in the rat:
involvement of both CB1 and CB2 receptors. J Neuroimmunol
2000;109:155–63.
a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 5 8 3 – 5 9 0590
41. Renzi A, De Luca Jr LA, Deróbio JG, Menani JV. Lesions of the
lateral hypothalamus impair pilocarpine-induced salivation
in rats. Brain Res Bull 2002;58:455–9.
42. Fernandez-Solari J, Prestifilippo JP, Vissio P, Ehrhart-
Bornstein M, Bornstein SR, Rettori V, et al. Anandamide
injected into the lateral ventricle of the brain inhibits
submandibular salivary secretion by attenuating
parasympathetic neurotransmission. Braz J Med Biol Res
2009;42:537–44.
43. Matsuo R, Kusano K. Lateral hypothalamic modulation of
the gustatory-salivary reflex in rats. J Neurosci 1984;4:
1208–16.
	Participation of the endocannabinoid system in lipopolysaccharide-induced inhibition of salivary secretion
	Introduction
	Materials and methods
	Chemicals
	Animals
	‘‘In vivo’’ studies
	Salivary secretion studies
	‘‘In vitro’’ studies
	Radioimmunoassay
	Anandamide production
	Determination of TNF&alpha;
	Statistics
	Results
	Effect of cannabinoid receptor antagonists and etanercept on LPS-induced inhibition of salivary secretion
	Effect of LPS administration on cAMP content and anandamide production in the submandibular gland
	Effect of LPS on TNF&alpha; plasma levels and content in the submandibular gland
	Inhibitory effect of TNF&alpha; on cAMP content in submandibular glands in vitro
	Effect of TNF&alpha; and cannabinoid receptor antagonists on salivary secretion
	Discussion
	Acknowledgements
	References